Accepted Manuscript

The combination of Nanomicelles with Terpenes for Enhancement of Skin DrugDelivery

Emine Kahraman, Neşet Neşetoğlu, Sevgi Güngör, Duri Şehvar Ünal, YıldızÖzsoy

PII: S0378-5173(18)30638-0DOI: https://doi.org/10.1016/j.ijpharm.2018.08.053Reference: IJP 17739

To appear in: International Journal of Pharmaceutics

Received Date: 4 July 2018Revised Date: 26 August 2018Accepted Date: 28 August 2018

Please cite this article as: E. Kahraman, N. Neşetoğlu, S. Güngör, D. Şehvar Ünal, Y. Özsoy, The combination ofNanomicelles with Terpenes for Enhancement of Skin Drug Delivery, International Journal of Pharmaceutics(2018), doi: https://doi.org/10.1016/j.ijpharm.2018.08.053

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The combination of Nanomicelles with Terpenes for Enhancement of Skin Drug Delivery

Emine Kahraman1, Neşet Neşetoğlu2, Sevgi Güngör1, Duri Şehvar Ünal2, Yıldız Özsoy1,*

1 Dept. of Pharmaceutical Technology, Faculty of Pharmacy, Istanbul University, Beyazıt, 34116, Istanbul,

Turkey

2 Dept. of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, Beyazıt, 34116, Istanbul, Turkey

* Corresponding author's e-mail: yozsoy@istanbul.edu.tr

Abstract

The nanomicelles have recently drawn a great deal of attention for drug delivery into the skin.

However, these carriers have only deposited in hair follicles and furrows, and drug in the micelles

may not therapeutically reach into viable skin layers. The aim of this study was to formulate a

combination of nanomicelles with terpenes to overcome this challenge and evaluate their potential

for topical drug delivery into the skin. The nanomicelles were characterised with respect to size, size

distribution (PDI), zeta potential, morphology and encapsulation efficiency (%). The drug

accumulation and penetration were examined by tape stripping method in the skin. The colloidal

stability of nanomicelles was followed with respect to size and PDI values. The nanomicelles were

about 25-30 nm in size with narrow distribution. All of them had slightly negative surface charge,

spherical shapes and high encapsulation efficiency (%). The tape stripping data revealed that

nanomicelles consisting of terpinolene led to accumulation of more drug in the stripped skin as

compared with commercial product and nanomicelles without terpene. Also, micelle formulations

consisting of terpinolene (2.0 %) had the highest colloidal stability. Consequently, combination of

nanomicelles with terpinolene could be a feasible approach for enhancement of skin drug delivery.

Keywords: nanomicelle, polymeric micelle, terpene, skin topical delivery, skin penetration

enhancement, penetration enhancer

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1. Introduction

The nanomicelles are aggregates with "core-shell" structures, self-assembling from amphiphilic

polymers at a specific polymer concentration and temperature in an aqueous medium (1, 2). The

"core" formed from hydrophobic polymer units has increased solubility of lipophilic drugs,

meanwhile, the "shell" has ensured physical stability of the micelles in an aqueous medium, helping

with steric hindrance in polyethylene glycol groups of hydrophilic polymer units (1-3). Although

these nano-carriers are mostly investigated for the treatment of cancer and inflammatory diseases in

the literature (2), some researchers suggested that an increment in the thermodynamic activity of

the compound in consequence of the increased drug solubility via the nanomicelles contributed to

enhancement of drug delivery into the skin (4-6). Thus, the nanomicelles have recently attracted a

great deal of attention for drug delivery into the skin for the treatment of dermatological diseases.

These carriers have only accumulated in the hair follicles and furrows, without passing the stratum

corneum barrier after applied on the skin. Afterward, the drug molecules have been released from

the micelles in the hair follicles and reached into the viable skin layers, partly overcoming the

stratum corneum barrier (4-6). However, drug amount released from the micelles could be

inadequate for effective treatment of the skin diseases because the hair follicles are less than 0.1 %

of the total skin surface in the human body.

The addition of penetration enhancers into topical formulations is an essential approach which is

often used to improve the penetration of drugs into the skin layers (7). The terpenes are generally

regarded to be safer in comparison with frequently-used penetration enhancers such as fatty

acids/esters, ethanol, urea, dimethyl sulphoxide, and surfactants (8). These excipients disrupt the

structure of the intercellular lipid layers in the stratum corneum to increase skin penetration of both

hydrophilic and lipophilic drugs (9, 10). However, they have been particularly useful for delivery of

small molecules (<500 Da) into the skin (11). Based on aforementioned knowledge, we aimed (i) to

develop an aqueous formulation for topical delivery of a lipophilic compound, which resulted in high

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patient compliance because of easy application and absence of greasiness, with drug content

equivalent to that of a commercial product, and (ii) to enhance delivery of a drug with high

lipophilicity and large molecular weight into the deeper layers of skin, utilizing the increase in drug

solubility via the nanomicelles and impairment of the stratum corneum through terpenes

2. Materials and Methods

2.1. Materials

Pluronic® F127 and L61 were kind gifts of BASF (Ludwigshafen, Germany). Tacrolimus monohydrate

(TAC) was kindly provided by Bilim Pharmaceutical Company (Turkey). D,l-limonene, d,l-menthol, and

nerolidol were obtained from Merck (Darmstadt, Germany). Terpinolene and eucalyptol were

purchased from Fluka (St. Gallen, Switzerland). All other chemicals and reagents were of analytical

grade and were commercially available.

2.2. Preparation of Nanomicelles

Tacrolimus monohydrate (TAC), which is a model drug, loaded polymeric micelles were prepared by

thin film hydration method (12). Briefly, Pluronic® F127 and Pluronic® L61 were dissolved in 1.5 mL of

acetonitrile. TAC was dissolved in 1.5 mL of acetonitrile. The solutions were mixed with each other. In

order to obtain a thin film layer in a round-bottom flask, acetonitrile was evaporated in a rotary

evaporator (IKA HB10 Control, IKA Works GmbH & Co., Germany) at 45°C, 150 mbar and 75 rpm/min.

Then, the thin film layer was kept overnight in a desiccator to remove completely the residual

acetonitrile. The film layer was hydrated with 3 mL of ultrapure water, stirring at 60°C for 1 hour.

After 1 hour, the size and size distribution of clear formulations were measured.

On the preliminary studies, micelle formulations were prepared with using different concentration of

polymers (1, 2, 3, 4, 5, 10 or 15 %, w/v of Pluronic® F127 and 0, 0.1, 0.25 or 0.5 %, w/v of Pluronic®

L61) and TAC (0.03, 0.05 or 0.1 % w/v) . In order to determine optimum concentration of polymers

and drug, the size stability of clear formulations was examined with DLS measurements for 14 days at

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room temperature, then the formulation exhibiting the highest colloidal stability was determined to

prepare the micelles consisting of penetration enhancer.

Different types of terpenes namely, d,l-limonene, terpinolene, d,l-menthol, eucalyptol or nerolidol as

penetration enhancers at the various of concentration (0.5, 1.0 or 2.0 %, v/v) were added into

micellar formulations. For the preparation of polymeric micelles containing penetration enhancer,

terpenes were added to mixed acetonitrile solution composed of polymer and drug, and the

procedure resumed as aforementioned before. The codes of obtained micellar formulations are

presented in Table 1.

Table 1: Formulation codes and terpenes (%, v/v) in the nanomicelles

Formulation Codes* Type of Terpenes % Terpenes (v/v)

TD401 - - - -

LD05 d,l-Limonene 0.5 - -

LD10 d,l-Limonene - 1.0 -

LD20 d,l-Limonene - - 2.0

TD05 Terpinolene 0.5 - -

TD10 Terpinolene - 1.0 -

TD20 Terpinolene - - 2.0

MD05** d,l-Menthol 0.5 - -

* All formulations contained 0.03 % of TAC, 4 % of Pluronic® F127 and 0.1 % of Pluronic ®L61

** (%, w/v)

2.3. Size and Size Distribution of Nanomicelles

The hydrodynamic size and size distribution (polydispersity index, PDI) of the micelles were

measured using dynamic light scattering (DLS) method by a ZetaSizer Nano ZS (Malvern Instruments,

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UK) at 25±0.1°C with angle of 173o. Each measurement was repeated at least three times for all

samples.

2.4. Zeta Potential of Nanomicelles

The zeta potential of the micelles was determined by a ZetaSizer Nano ZS (Malvern Instruments, UK)

at 25±0.1°C, using electrophoretic light scattering method. The time and voltage parameters were

automatically set. The measurement parameters were as follows; refractive index: 1.330; viscosity:

0.8872; dielectric constant of water: 79 and f(ka):1.50 (Smoluchowski value). Each measurement was

repeated at least three times.

2.5. Morphology of Nanomicelles

The morphology of the micelles was observed by Atomic Force Microscopy (SPM-9600, Shimadzu,

Japan). A sample of the micellar formulation was fixed onto the mica surface after the mica surface

was cleaned. The resonance frequency and force constant were set to 320 kHz and 42 N/m,

respectively. Then, the sample was scanned in "dynamic mode".

2.6. Encapsulation Efficiency and Drug Loading of Nanomicelles

Vivaspin®6 centrifuge tubes with polyethersulphone membrane (MWCO: 3500 Da, Sartorius AG,

Germany) were used to determine encapsulation efficiency and drug loading capacity of the

nanomicelles (13). 3 mL of the micellar formulation was placed into the centrifuge tube and

centrifuged for 30 minutes at 3000 RCF (g). 100 µL of filtrate was diluted with 0.9 mL of acetonitrile

and concentration of TAC in the samples was quantified by validated High Pressure Liquid

Chromatography (HPLC) as described below. The encapsulation efficiency (%) and drug loading

capacity (%) of the micelles were estimated according to Eqs (1) and Eqs (2), respectively;

Encapsulation Efficiency (%)=100-(ax100/b) (1)

Drug Loading Capacity (%)=[(b-a)/(b+c+d)]x100 (2)

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a: The concentration of TAC in filtrate (mg/mL)

b: The concentration of TAC used for micellar formulation (mg/mL)

c: The concentration of Pluronic® F127 (mg/mL)

d: The concentration of Pluronic® L61 (mg/mL)

2.7. Skin Accumulation and Penetration Studies

2.7.1. Skin Preparation

The excised porcine skin was used as a model membrane for the skin accumulation and penetration

studies. The porcine dorsal skin was obtained from a local slaughterhouse, washed carefully under

cold running water and blotted dry with soft tissue. The hairs were cut with a scissors and

subcutaneous fatty tissue was carefully removed using a scalpel from the skin. After that, the skin

was packed in aluminium foil and frozen at -20°C until use. The skin samples were thawed at room

temperature before the experiments and the skin surface was carefully cleaned with cold phosphate-

buffered saline (pH 7.4, PBS).

2.7.2. Skin Permeation Studies

The skin permeation studies were performed using Franz diffusion cells (PermeGear, V6A Stirrer,

Hellertown, PA, USA) with an available diffusion area of 1.77 cm2 and a receptor volume of 12 mL.

The receptor chambers were filled with PBS (pH 7.4) containing serum bovine albumin (BSA) of 1 %

(w/v) to maintain the sink condition during the experiment (5). The stratum corneum layer of full-

thickness skin samples was exposed to donor compartments and mounted onto Franz diffusion cells,

the donor chambers were clamped. The receptor medium was magnetically stirred at 300 rpm and

kept at 37±0.5 °C during the experiment, to reach 32±0.5 °C for the skin temperature. All air bubbles

in the receptor medium were carefully removed. After equilibration for 30 min, 1 mL of the micellar

formulations and 1 g of commercial ointment (Protopic®, 0.03 %) as control formulations were

applied to the skin surface in the donor compartments. At the end of 24 hours, 1 mL of samples were

7

withdrawn from the receptor chambers, filtered through 0.45 µm Millex® syringe filters (PTFE, LCR,

Merck Millipore, Darmstadt, Germany). The drug concentration in the samples was quantified by

LC/MS using the method described below.

2.7.3. Skin Cleaning and Tape-Stripping Prodecures

Upon completion of skin permeation studies, the receptor medium was removed and the diffusion

cells were dismantled. The skin surface was washed 5 x 1 with mL ultra pure water in order to

remove the remaining formulation and then, was gently dried with a cotton swab (14).

The tape stripping method was used to separate the stratum corneum from the viable epidermis and

dermis (15, 16). To avoid any furrows which could result in mistaken data of the tape stripping

procedure, the skin was stretched and mounted with pins on cork disks. 20 strips (2x2 cm2) of

transparent adhesive tape (3M, Hutchinson, MA, USA) were sequentially fixed on the cleansed skin.

In order to ensure a reproducible working procedure, a constant pressure was applied on the skin

moving a roller (400 g) for ten seconds and the tape was removed from the skin surface in a single

rapid movement at an angle of 45°. This template ensured for all tape stripping procedures at the

same skin specimen. The first tape was discarded to represent unabsorbed drug in the skin surface

and the other sequential tape strips were placed in four vials (4 strips in the first vial, 5 strips in the

other vials) of suitable size for the extraction. The remaining skin samples were also cut into smaller

pieces to increase the surface area and placed in a separate vial. To dissolve the drug in the

extraction fluid, each of the samples (stratum corneum or stripped skin) was soaked in acetonitrile (4

x 6 mL per vial for tape strips, 4 mL for the stripped skin samples) at ambient temperature for 15

hours under constant shaking (Thermo Forma 420 Orbital Shaker, Waltham, MA, USA) and vortexed

vigorously for 5 minutes. The samples were filtered through 0.45 µm Millex® syringe filters, 0.5 mL of

each tape strip sample was collected and mixed for 2 minutes. Then, all of the samples was

submitted to LC/MS analysis for quantification of drug content.

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2.8. Quantification of Drug Amounts by HPLC and LC-MS/MS Analysis

2.8.1. HPLC Analysis

TAC concentration in the samples was quantified by HPLC equipped with PDA detector (LC 20AT,

Shimadzu, Japan). C18, 250 mm x 4.6 mm, i.d. 5 µm column (Thermo Scientific, USA) was used as

stationary phase. The mobile phase was a mixture of acetonitrile: water: phosphoric acid

(750:250:0.2, v/v/v) filtered through membrane filter (0.45 μm, Millex LH, Merck Millipore,

Germany). The analysis was performed at 215 nm of detection wavelength and 1.0 mL/min of flow

rate. The injection volume was applied as 50 µL. The method was validated for selectivity, linearity,

accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). The validation

parameters were found to be linear in concentration range of 0.5 µg/mL and 100 µg/mL (r2 > 0.999),

accurate (recovery > 98%), precise (intra and inter day variation < 2 %). LOD and LOQ values were

0.094 µg/mL and 0.286 µg/mL, respectively. Any peaks of the polymers and penetration enhancers

did not interfere with TAC peak, demonstrating selectivity of the analytical method.

2.8.2. LC-MS/MS Analysis

In order to improve specificity and sensitivity, LC-MS/MS analysis was carried out to quantify the

drug in the penetration studies by LC/MS system (Agilent Technologies 6460 Triple Quadrupole,

California, USA) comprising Infinity 1260 series binary pump and jet stream electrospray ionization.

C18, 150 mm x 4.6 mm, i.d. 5 µm analytical column (Thermo Scientific, USA) was used for isocratic

separation at 25°C. The mobile phase consisted of methanol: ultra pure water with 1.0 % of formic

acid (99:1, v/v). The flow rate was set at 0.7 mL/min and injection volume was applied as 2 µL.

Repaglinide (REP) at a concentration of 50 ng/mL was used as internal standard. Mass spectrometric

detection was performed with electrospray ionization in positive ion mode using multiple reaction

monitoring. The detection settings for TAC and REP are presented in Table 2. The method was

validated for selectivity, linearity, accuracy and precision. The linear range during the measurements

for TAC was from 2 to 500 ng/ml (r2 > 0.999).

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Table 2: LC/MS settings for detection of tacrolimus and repaglinide

Tacrolimus Repaglinide

Source parameters Value (+) Value (-)

Polarity Positive Positive

Precursor ion (m/z) 826.4 453.4

Product ion (m/z) 616.3 162.3

Collision energy (V) 36 15

Fragmentor voltage (V) 180 120

Cell accelerator voltage (V) 7 7

Capillary voltage (kV) 3.50 3.50

Nozzle Voltage (V) 500 500

Drying gas temperature (°C) 300 300

Drying gas flow (L/min) 5 5

Nebulizer (psi) 45 45

Sheat gas heater (°C) 250 250

Sheat gas flow (L/min) 11 11

2.9. Colloidal Stability of Nanomicelles

The colloidal stability of the micelles was examined at room storage conditions (25±2°C, 60±5%

relative humidity (RH)) and, at accelerated stability test conditions (40±2°C, 75±5% RH), in

accordance with ICH Q1 (R2) Guideline. The hydrodynamic size and size distribution of polymeric

micelles were measured at the end of 15th day, 1st month, 2nd month and 3rd month by ZetaSizer

Nano ZS (Malvern Instruments, UK) following macroscopically observation. Each measurement was

repeated at least three times.

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2.10. Data Analysis

The obtained data are presented as mean of three experiments ± standard deviation (SD). All of the

data was assessed with Unpaired Student’s t-test or One-way ANOVA, followed by the Bonferroni

multiple comparison test, using GraphPad Prism Software version 6.05VR (La Jolla, USA). P value

<0.05 was considered as level of statistical significance.

3. Results and Discussion

3.1. Preparation of Nanomicelles

Pluronic® micelles recognizing as safe have been intensively studied for a long time, despite their low

physical stability (17-20). However, their colloidal stability and drug loading capacity could be

increased with a mixture of Pluronic® copolymers. Alakhov et al. reported that Pluronic® F127 (2 %)

and Pluronic® L61 (0.25 %) mixed micelles were stable in the presence of fetal bovine albumin (2.5 %)

(21). Based on this information, the mixture of Pluronic® F127 and Pluronic® L61 was also used to

maintain colloidal stability of the micelles in our study. On the preliminary studies, Pluronic® F127

was worked in concentration range of 1 % (w/v) and 15 % (w/v), because of 0.8 % (w/w) < critical

micelle concentration (22) and 18 % (w/w) < gelation concentration at body temperature (23).

However, Pluronic® F127 concentration was selected between 1 % (w/v) and 5 % (w/v) to form the

micelles because more than 5 % (w/v) of polymer concentration gelled in the hydration process.

Pluronic® L61 was added to the formulations at three different concentration (0.1, 0.25 and 0.5 %,

w/v) based on the literature (21).

The drug concentration loading into the micelles has affected their formation, size and colloidal

stability (24, 25). At the end of 14 days, some visible aggregates were viewed in the blank

formulations (the micelles not loading TAC) and the most of drug loaded micelles. Conversely, the

micelles with drug concentration of 300 µg/mL showed higher colloidal stability from blank and more

drug loaded micelles. This situation indicated that drug loading in the micelles improved colloidal

11

stability, but there has been a saturation limit of the drug as pointed out by Zhang et al (26). Overall,

TD401 coded formulation had the smallest size distribution value (0.218 ± 0.085) for 14 days and its

content was used for further studies.

To the best of our knowledge, excipients have also affected micelle formation and characterisation

(27, 28). Based on this challenge, we need to study with five different types of terpenes for a well-

designed formulation. In our study, the presence of nerolidol, eucalyptol and menthol (except of 0.5

%, w/v) in the formulation prevented formation of the micelles, unlike limonene and terpinolene.

When considered of log P values (29-31), molecular formulas of terpenes and the other components

(Figure 1), it could be claimed that a balance of hydrophilic and lipophilic groups might need for

micelle formation and log P values of terpenes should be in a certain range to form the micelles.

“INSERT FIGURE 1”

3.2. Size and Size Distribution of Nanomicelles

The particle size has played an important role for drug delivery into the skin. Campbell et al. (32)

reported that polymeric particles accumulated only on the skin surface despite their small size (20

nm), without reaching into the viable layers. Vogt et al. (33) also indicated that 40 nm particles

penetrated to follicular epithelium, but larger particles localised in entry of hair follicles. Therefore,

the micellar size is a crucial parameter in view of predicting where they deposit in the skin. In the

study, all of the micellar formulations were in range of 25 nm and 35 nm (Table 3). The presence of

terpinolene or menthol in the formulations decreased micellar size and caused to an unimodal

distribution (Table 3) in comparison with the micelles with limonene and the without terpene.

Particularly, the formulation containing terpinolene (TD20) exhibited significantly smaller size and

narrower size distribution than control formulation without penetration enhancer (TD401) (p<0.05).

When the concentration of TAC in formulations with terpinolene were increased to 1000 µg/mL, it

was observed that both of the size and size distribution was not significantly changed (p>0.05) (data

12

not given). Hence, it could be claimed that terpinolene could reduce micellar size and size

distribution, independently of TAC concentration. Furthermore, menthol caused to a significant

decrease in size of the micelles only in which the drug concentration was 300 µg/mL (p<0.05). In the

micelles with limonene, addition of terpenes into the formulation resulted in increased size and size

distribution value of the micelles loaded TAC at concentration of 300, 500 or 1000 µg/mL (data not

given). This could depend on intensity of micelles with increasing of limonene concentration, unlike

ones with terpinolene and menthol (Figure 2).

Table 3: The size, size distribution and zeta potential of the nanomicelles

Formulation

Codes

Z-Average Size

(d. nm) ± SD* PDI ± SD

Size Range

(nm)**

Zeta Potential

(mV) ± SD

TD401 28.85 ± 1.36 0.263 ± 0.040 (Bimodal) 10-16 -2.02 ± 0.56

LD05 29.37 ± 1.40 0.259 ± 0.006 (Bimodal) 15-20 -1.73 ± 0.93

LD10 30.28 ± 2.30 0.325 ± 0.093 (Bimodal) 15-24 -2.26 ± 1.19

LD20 34.70 ± 2.18 0.425 ± 0.096 (Bimodal) 15-29 -2.69 ± 0.83

TD05 29.77 ± 7.94 0.225 ± 0.053 (Unimodal) 13-19 -3.92 ± 0.67

TD10 27.73 ± 4.61 0.215 ± 0.052 (Unimodal) 5-39 -3.30 ± 1.00

TD20 25.76 ± 0.84 0.152 ± 0.015 (Unimodal) 14-16 -2.28 ± 1.11

MD05 25.32 ± 0.58 0.187 ± 0.065 (Unimodal) 5-27 -2.93 ± 2.17

* DLS data

** AFM data

3.3. Zeta Potential of Nanomicelles

The surface charge of nanoparticles may act as a crucial factor in terms of their colloidal stability and

deposition in the skin (34, 35). Hence, we measured zeta potential values of the micelles to comment

13

the colloidal stability and deposition in the skin against the surface charges. The zeta potential values

were in the range of -1.73 ± 0.93 and -3.92 ± 0.67 mV (Table 3), with respect to the nature of the

copolymers and no significant differences existed between the micellar formulations comprising

terpene and control formulation (TD401) (p>0.05). As a result, these findings showed that the

colloidal stability and dermal deposition would not be affected by surface charges of the micelles, in

the following experiments.

3.4. Morphology of Nanomicelles

AFM images have revealed that all of micellar formulations consisting of terpenes had spherical

shape (Figure 2) as generally seen in Pluronic® micelles (36, 37). Therefore, it may be claimed that

the monoterpenes such as d,l-limonene, terpinolene etc. did not prevent micelle formation and/or

disrupt their spherical shapes. The size of micelles consisting of terpenes from AFM images was

smaller than those of measured with ZetaSizer (Table 3). In ZetaSizer measurements, this

phenomenon could regard to calculation of diffusion layer formed due to H+ and OH- ions in the

water (38, 39). Interestingly, it was also observed in AFM images that adsorbed the micelles

intensified on the mica surface when concentration of the terpene increased in the micellar

formulations with limonene, unlike terpinolene.

“INSERT FIGURE 2”

3.5. Encapsulation Efficiency and Drug Loading of Nanomicelles

Pluronic® micelles differing from their terpene content (from 0.5 up to 2 %, v/v) were formulated and

characterised (Table 3 and Figure 2). Encapsulation efficiency (%) and drug loading efficiency (%)

increased for all micellar formulations containing any terpene (99.0 % < and approximately 0.73) as

compared to control micelles without terpene (94.91 % ± 0.88 and 0.69 ± 0.01), respectively. In

addition, drug concentration in control formulation (TD401) was 15.27 ± 2.60 µg/mL in the filtrate,

whereas no drug was detected in the filtrate of the formulations containing terpene, indicating an

14

increase in drug loading capacity of the micelles with the presence of terpenes. The literature also

affirmed that TAC is slightly soluble in water (4-18 μg/mL) despite being a lipophilic compound (40,

41). This was attributed to dissolution of the drug in the terpenes which interacted with

polypropylene core of Pluronic® polymers and increased the solubility of TAC in the core. Similarly,

Badran et al. (42) reported that the terpenes such as cineole increased encapsulation efficiency of a

hydrophobic drug (nimesulide) in the liposomes. Charoenputtakun et al. also revealed that limonene

increased solubility of all-trans retinoic acids, and then it was resulted in higher drug loading capacity

in the lipid nanoparticles (43).

3.6. Skin Accumulation and Penetration Studies

The efficiency of drugs applied topically depends on the concentration in targeted tissues, which is

related to penetration ability of drugs into the skin. Therefore, there is a need for targeting of drugs

into the skin layers since conventional dosage forms such as ointments could be inefficient to achieve

the required drug concentrations in the target cutaneous tissues. Hence, development of novel

carriers would have superiorities in terms of enhancement of therapeutic aspect (44).

In order to examine the skin penetration of TAC which has highly lipophilic structure (log P=3.96)

with large molecular weight (802.02 g/mol) via the nano-micelles consisting of terpenes, the

quantification of TAC in the stratum corneum was performed by sequential tape stripping method

following in vitro skin permeation study at infinite dose. For the comparison, the penetration of TAC

from its commercial ointment (Protopic®) and the terpene-free micelles was also examined. (Figure

3). After in vitro permeation study for 24 hours, no drug was detected in receptor fluids of the

diffusion cells applied either micelle formulations or Protopic ointment. When the micelles without

terpene (TD401) was compared with Protopic ointment, the micelles showed an increase in the

concentration of TAC by 4.2 fold in the stratum corneum and 3.2 fold in the total skin. This

enhancement could arise from binding of polyethylene glycol (PEG) (which is a hydrophilic polymer

15

and generates the shell of the micelles) to the water molecules and increasing hydration of the

stratum corneum. Similarly, Rangsimawong et al. also (45) reported that PEG could affect the

penetration of hydrophilic drug in the liposomes into the skin layers. TAC in the micelles without

terpen and conventional formulation accumulated at similar amount in the stripped skin. This may be

low diffusion rate of tacrolimus into the epidermis due to its large molecular weight and highly

lipophlicity. When applied the micelles containing d,l-limonene or terpinolene into the skin, drug

concentration in the stratum corneum were detected similar to that of the micelles without terpene

(TD401). However, TAC significantly accumulated much more in the stripped skin (p<0.05) following

the application of LD20, TD05, TD10 and TD20 micellar formulation. This may be due to stronger

penetration enhancer effect of d,l-limonene or terpinolene. In this instance, the micelles could have

localized in the hair follicles and furrows as reported in previous study (4) when applied onto the

skin. Meanwhile, the drug and terpene were released into the hair follicles and furrows, the terpene

broke the hydrogen bonds among the lipids in the stratum corneum and the drug penetration dually

increased. Additionally, the micelles could improve penetrability of the drugs by increasing its

thermodynamic activity and acting the penetration enhancer compound (5, 46). Interestingly, when

compared with the micelles without terpene (TD401), tacrolimus via the micelles with d,l-menthol

significantly penetrated more into the stratum corneum, conversely in the stripped skin. This may be

due to its poor colloidal stability.

Note that the finite dose which described as <10 μl/cm2 for liquids and 2-5 mg/cm2 for semi-solid

preparations in the donor compartment could not be applied in the penetration studies due to the

challenge of developing a bioanalytical method for tacrolimus in the skin studies. When considered

into the comparison of finite dose and infinite dose applications in guidance of earlier reported

studies (14, 47), it might be remarked that the absolute drug amounts delivered into the skin could

be lower from these results for the micellar formulations and commercial product if a finite dose was

applied in our study. The relative amounts recovered were conversely able to be higher for finite

16

dose studies. Furthermore, a finite dose of micellar formulations applied in the donor compartment

might cause to a decrease in hydration of the stratum corneum and reducing in drug penetration into

the skin as also reported by (14). On the other hand, this phenomena which is more possible for

hydrophilic drugs, could not significantly affect or might increase our results in comparison with the

commercial product because of difficulty the partition of highly lipophilic molecules into the

"hydrated" stratum corneum.

“INSERT FIGURE 3”

3.7. Colloidal Stability of Nanomicelles

The colloidal stability of the micelles with terpenes and control micelles storing at 25±2°C, 60±5% RH

and 40±2°C, 75±5% RH are presented in Figure 4. The hydrodynamic size and PDI results revealed

that terpinolene (especially at the concentration of 2.0 %, v/v) created a great effect on the colloidal

stability of the micelles. This increased stability could be explained by two mechanisms. Firstly, the

terpenes in liquid form may enable a more fluidic core in the micelles at room temperature when

localized in the hydrophobic units of the micelles. Similarly, Adams et al. showed that the presence of

the fluid core in the micelles affected on the kinetic and thermodynamic stability of these

nanocarriers (48, 49). Secondly, it has well known that an increase of the core lipophilicity has

resulted in a decrease of the critical micelle temperature (CMT), and then the improved kinetic

stability (50). As seen in our study, highly lipophilic terpinolene or d,l-limonene localizing in the

micelle core could reduce CMT of Pluronic® F127 and L61 which are thermo-sensitive polymers and

increase the stability. However, it could be suggested considering the similarity of molecular

structures of terpinolene and d,l-limonene (Figure 1) that the melting point and indirectly solubility

of the terpenes in the water affected the colloidal stability of the micelles. As seen in Figure 1, the

melting point of d,l-limonene is much lower than that of terpinolene. According to our knowledge,

the melting point of compounds is inversely proportional with the solubility. In this situation, it could

be asserted that the solubility of d,l-limonene in the water is higher than that of terpinolene and

17

hence, d,l-limonene could resulted in a higher affinity into the water phase than terpinolene, leakage

of d,l-limonene from the core in course of time and shorter colloidal stability period than the micelles

with terpinolene (Figure 4).

“INSERT FIGURE 4”

4. Conclusion

The combination of nanomicelles with terpenes was efficiently formulated and characterised in

terms of hydrodynamic size, size distribution, zeta potential and morhology. The spherical micelles

had slightly negative surface charge and high encapsulation efficiency (%) owing to terpene

molecules in the micelle core. The penetration studies revealed that the nanomicelles consisting of

terpinolene significantly increased the accumulation of a highly lipophilic and relatively large

molecule (TAC) into the viable epidermis and dermis as compared to the nanomicelles without

terpene and the commercial product. Depending on the terpene concentration, terpinolene

improved the colloidal stability of Pluronic® micelles at room storage conditions and at accelerated

stability test conditions. This study confirms that the combination of nanomicelles with terpenes are

an innovative carrier able to enhance topical drug delivery into the skin. Based on these positive

results, the penetration mechanism of these nanomicelles into the skin, cytotoxicity and biological

activity in the cell culture studies will be examined in the future studies.

Acknowledgements: This work was supported by the Research Fund of Istanbul University [Project

numbers: 50744].

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Legends for Figures

Figure 1: The chemical structures and physicochemical properties of tacrolimus (A) and the terpenes,

eucalyptol (B), nerolidol (C), d,l-limonene (E), terpinolene (E) and d,l-menthol (F)

Figure 2: AFM imagines of the nano-micelles A) without terpene (TD401), B) with 0.5 % of d,l-

limonene (LD05), C) 1.0 % of d,l-limonene (LD10), D) 2.0 % of d,l-limonene (LD20), E) 0.5 % of

terpinolene (TD05), F) 1.0 % of terpinolene (TD10), G) 2.0 % of terpinolene (TD20) and H) 0.5 % of

d,l-menthol (MD05)

Figure 3: Tacrolimus accumulation in the stratum corneum, the stripped skin and total skin through

commercial formulation (Protopic® Ointment) and the micelles

Figure 4: Change of hydrodynamic size (Z-average) and polydispersity index (PDI) of the nano-

micelles stored a) at 25◦C and b) 40◦C over 3 months

TACROLIMUS

TERPINOLENE

+

PLURONIC

MICELLE FORMATION

+

*Graphical Abstract (for review)

Log P Molecular Weight Melting Point

3.96 804.02 g/mol

27-129°C

2.82 154.3 g/mol

1.5°C

5.32 222.4 g/mol

- 75°C

Log P Molecular Weight Melting Point

4.58 136.2 g/mol

-74°C (d-limonene), -90°C (l-limonene)

4.52 136.2 g/mol

< 25°C

3.20 156.3 g/mol

- 8 °C (d-menthol), 41-43°C (l-menthol)

A B C

F E D

Figure 1

A

G F E

D C B

H

200 nm

Figure 2

0.0

0.1

0.2

0.3

0.4

*****

Protopic Ointment

TD401

LD05

LD10

LD20

TD05

TD10

TD20

MD05

*

Dru

g ac

cum

ulat

ion

in th

e st

ripp

ed sk

in (µ

g/cm

2 )

0

1

2

3

4

5

6 ****

Dru

g ac

cum

ulat

ion

in th

e to

tal s

kin

(µg/

cm2 )

0

1

2

3

4

5

6**

**

Dru

g ac

cum

ulat

ion

in th

estr

atum

cor

neum

(µg/

cm2 )

Figure 3

25oC

0 15 3020

25

30

35

40

Days after preparation

Hyd

rody

nam

ic s

ize (n

m)40oC

0 15 30 60 9020

25

30

35

40TD401LD05LD10LD20TD05TD10TD20MD05

Days after preparation

Hyd

rody

nam

ic s

ize

(nm

)

0 15 30

0.2

0.3

0.4

0.5

Days after preparation

Poly

disp

ersi

ty in

dex

(PD

I)

0 15 30 60 900.1

0.2

0.3

0.4

0.5

0.6

Days after preparation

Poly

disp

ersi

ty in

dex

(PD

I)

Figure 4